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Updated: Feb 2, 2026

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry
Published on: July 20, 2016
Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins
Dennis Szymanski1, Malvina Papanastasiou1, Lakshmipathi Pandarinathan1
1Center for Drug Discovery , Northeastern University , 116 Mugar Hall, 360 Huntington Avenue , Boston , Massachusetts 02115 , United States.
Abstract:
Upon ultraviolet activation, cannabinergic aliphatic azido (N3) ligands covalently label cannabinoid receptors, prominent G-protein-coupled receptor (GPCR) drug targets. We report here the mechanism of covalent attachment to selected substrates of the high-affinity CBR inverse agonist AM1335 and its deuterated analog AM1335(d10), arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6) peptide and an N-acyl-protected cysteine (NAC). The photochemical reaction products of model substrates with AM1335 and AM1335(d10) were analyzed with tandem electrospray ionization mass spectrometry fragmentation and deuterium exchange mass spectrometry. The nitrene initially formed after photoreaction undergoes rearrangement to an imine which then interacts with the cysteine sulfhydryl group, resulting in ligand attachment. Our results demonstrate that covalent probes carrying aliphatic azides behave more selectively than originally thought and can be used to label protein cysteine residues preferentially.
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